Cross-linked polysaccharide drug carrier

ABSTRACT

A carrier and a method for preparing it are provided for use in the delivery of therapeutic agents. A polysaccharide is reacted with an oxidizing agent to open sugar rings on the polysaccharide to form aldehyde groups. The aldehyde groups are reacted to form covalent oxime linkages with a second polysaccharide and each of the first and second polysaccharide is selected from the group consisting of hyaluronic acid, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate and alginate.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/110,381, filed Jul. 1, 1998, which is a continuation-in-part of U.S.application Ser. No. 08/887,994, filed Jul. 3, 1997.

BACKGROUND OF THE INVENTION

[0002] The present invention is directed to biodegradable carriers forthe delivery of therapeutic agents, methods of making the carriers andmethods of using the carriers.

[0003] There is a clinical demand for carriers of therapeutic agentsthat are biodegradable, biocompatible and which allow for targeteddelivery and controlled release of the therapeutic agent.

[0004] Polysaccharides, such as hyaluronic acid (HA) and dextran sulfatehave been used in a wide variety of biomaterials. Hyaluronic acid (HA),a naturally occurring polysaccharide, has been used in matrixengineering in ophthalmic and orthopedic medicine. Clinical indicationsfor HA alone are limited by its physical properties and the shortresidence time of the natural HA molecule. A formaldehyde cross-linkedHA, Hylan, has been used in viscosupplementation of arthritic diseasedjoints (Takigami et al., 1993, Carbohydrate Polymers 22: 153-160).Dextran sulfate, a glycosaminoglycan-like polyionic derivative ofdextran, has been shown to be useful as a biomaterial and drug fortreatment of hyperlipidemia. It is produced by esterification ofdextran, a hydrophilic polymer of glucose synthesized by certain strainsof bacteria.

[0005] Berg et al., (U.S. Pat. No. 5,510,418, issued Apr. 4, 1996)disclose glycosaminoglycans, such as, HA, chondroitin sulfates, keratansulfates, chitin and heparin, chemically conjugated to a synthetichydrophilic polymer, such as polyethylene glycol (PEG) that are used asinjectable formulations or solid implants. Koji Kimata et al., (U.S.Pat. No. 5,464,942 issued Nov. 7, 1995) disclose phospholipid linkedglycosaminoglycans and their use as metastasis inhibitors. Sakurai, etal, U.S. Pat. No. 5,310,881 issued May 10, 1994, discloseglycosaminoglycan-modified proteins. Balazs et al., U.S. Pat. No.5,128,326 issued Jul. 7, 1992, disclose hyaluronan cross-linked withdivinyl sulfone.

SUMMARY OF THE INVENTION

[0006] The present invention provides biodegradable carriers for thedelivery of therapeutic agents, methods of making the carriers andmethods of using the carriers.

[0007] A biodegradable carrier of the present invention comprises across-linked first and second polysaccharide, wherein each of the firstand the second polysaccharide is a derivative of a member selected fromthe group consisting of hyaluronic acid, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparansulfate and alginate. The first polysaccharide contains aldehyde groupsderived from oxidized sugar rings. The second polysaccharide an aminederivative and the first and second polysaccharides are covalentlycross-linked through these groups which forms imine linkages. In thepresent invention, the cross-linking reaction proceeds without utilizingextraneous cross-linking or ionic binding agents.

[0008] The method of making the biodegradable carriers comprises thesteps of oxidizing a first polysaccharide to form a first polysaccharidederivative having aldehyde groups, and reacting the first polysaccharidederivative with a second polysaccharide amine derivative underconditions such that the aldehyde groups covalently react with the aminesites to form a cross linked carrier.

[0009] The present invention also provides methods of using the carrierto deliver therapeutic agents by administering the carrier at the sitesof desired therapeutic intervention.

[0010] The ratios of the first and second polysaccharide can be variedto change both the physical and biological properties of the carrier.For example, a higher ratio of aldehyde bearing polysaccharide would bepreferred for immobilizing a therapeutic agent to the carrier. Thepresence of unreacted but active aldehydes provides sites for covalentlinkage to a therapeutic agent.

[0011] A carrier of the present invention can be produced in a varietyof physical forms. For example, it can be made into a gel-like form forinjection or a sponge-like form for implantation at a desired site oftherapeutic intervention.

[0012] A carrier of the present invention provides the advantage ofbeing biocompatible while maintaining a prolonged biodegradation ratedue to the cross-linking; providing controlled release of thetherapeutic agent and having the flexibility of formulation in gel-likeor sponge-like form to accommodate desired therapeutic intervention.

[0013] As used herein therapeutic agent means any bioactive agent, suchas a protein, polypeptide, or amino acid, including growth factors,growth factor receptors, cytokines, hormones, antibodies or chemicalagents, such as, for example, non-peptide hormones chemical mimetics ofgrowth factors and receptors that have been shown to have a biologicaleffect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 illustrates the release of crystal violet encapsulatingwithin a carrier of the present invention.

[0015]FIG. 2 illustrates the release of FITC-BSA immobilized within acarrier of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The method of preparing a carrier of the present inventioncomprises the steps of opening sugar rings on a first polysaccharide andoxidizing terminal hydroxy groups to aldehydes using, for example,sodium or potassium periodate as a selective oxidizing agent. The amountof aldehyde groups produced in this manner can be stoichiometricallycontrolled. Typically, from about 1% to 50% of the rings can be openedin this manner. More preferably about 1% to 10% of the repeat sugarunits are opened to form aldehyde groups. These aldehyde groups can formcovalent imine crosslinks with the second polysaccharide aminederivative at amine sites. The reagents for opening sugar rings on thefirst polysaccharide may be any selective oxidizing agent which oxidizesa terminal hydroxyl group to an aldehyde, including specific sugaroxidases.

[0017] In the present invention the first and second polysaccharides areeach selected from the group consisting of hyaluronic acid, dextran,dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate,heparin, heparan sulfate and alginate. In a preferred embodiment, thefirst and second polysaccharides are each selected from the groupconsisting of hyaluronic acid and chondroitin sulfate. As used herein,the term polysaccharide includes the polysaccharide and its salts suchas the sodium, potassium, magnesium, calcium, and the like, salts.Preferred forms of starting material of the polysaccharides includethose which have been approved for human use. The starting material forhyaluronate can be derived by bacterial fermentation or throughisolation from rooster combs or can be obtained from commercial sources.

[0018] The carrier may be comprised of the same or different first andsecond polysaccharides. In one preferred embodiment, the first andsecond polysaccharide are both hyaluronic acid. In another preferredembodiment, one polysaccharide is HA and the other is chondroitinsulfate. Typically, the polysaccharides will have an average molecularweight of about 1,000 to 10,000,000 DA.

[0019] A carrier of the present invention may be formulated in severalphysical forms, including gel-like or sponge-like forms. When it isdesirable to obtain sustained or slow release delivery of thetherapeutic agent, the agents may be immobilized by attachment to thecarrier. A carrier gel, sponge, or microparticle preparation can beprepared by using a polysaccharide polyaldehyde derivative in excess,resulting in a carrier having unreacted, while still active aldehydeswhich are suitable for the immobilization of free amine containingbioactive therapeutic agents. Proteins and many growth factors are freeamine-containing compounds.

[0020] Where it is desirable to achieve short-term delivery of atherapeutic agent, the agent may be entrapped within the carrier. Drugs,growth factors, polypeptides, proteins, and other bioactive therapeuticagents can be entrapped within the gel/sponge either by mixing the agentwith one of the two derivatives before gelatinization, or diffusion froma drug solution into the gel/sponge after their formation.

[0021] The agent may also be covalently linked to the carrier, forexample, via an imine bond. Some of the aldehyde groups on the carrier,prior to forming a gel or sponge, may be reacted with amine groups onthe agent.

[0022] The carrier may be formulated into a gel-like carrier when it isdesirable to produce an injectable formulation, which can be deliveredarthroscopically, or by syringe or catheter. Alternatively, the carriermay be formulated into a sponge-like carrier when it is desirable toproduce an implantable formulation. The carriers of the presentinvention may be formed into any shape by lyophilization or air dryingin molds of the desired shape. The lyophilized material may also beformed into a viscous gel by rehydrating the lyophilized material.

[0023] Examples of therapeutic agents which may be used in the presentinvention are not particularly restricted and include proteinsoriginating from various animals including humans, microorganisms andplants as well as those produced by chemical synthesis and using geneticengineering techniques. Therapeutic agents include, but are not limitedto, growth factors such as, bFGF, aFGF, EGF (epidermal growth factor),PDGF (platelet-derived growth factor), IGF (insulin-like growth factor),TGF-β 1 through 3, including the TGF-β superfamily (BMP's, GDF-5, ADMP-1and dpp); cytokines, such as various interferons, includinginterferon-alpha, -beta and -gamma, and interleukin-2 and -3; hormones,such as, insulin, growth hormone-releasing factor and calcitonin;non-peptide hormones; antibiotics; anti-cancer agents and chemicalagents, such as, chemical mimetics of growth factors or growth factorreceptors, and DNA constructs, including cDNA constructs and genomicconstructs). In a preferred embodiment, the therapeutic agents includethose factors, proteinaceous or otherwise, which are found to play arole in the induction or conduction of growth of bone, ligaments,cartilage or other tissues associated with bone or joints, such as forexample, BMP and bFGF. The present invention also encompasses the use ofautologous or allogeneic cells encapsulated within the carrier. Theautologous cells may be those naturally occurring in the donor or cellsthat have been recombinantly modified to contain nucleic acid encodingdesired protein products.

[0024] As will be understood by those of skill in the art, the amount oftherapeutic agent to be immobilized or encapsulated within the carrierwill vary depending upon the intended therapeutic target, but willusually be in the range of picogram to gram quantities.

[0025] A carrier of the present invention may be administered throughimplantation, direct application or injection depending on the intendedtherapeutic application, the physical properties and the ratio ofpolysaccharide derivatives.

[0026] The efficacy of therapeutic delivery of such agents can be shownby both in vitro and in vivo tests known by those of ordinary skill inthe art. In the present invention, the preferred therapeutic agents arethose factors which are found to play a role in the induction orconduction of growth of bone, ligaments, soft tissue, cartilage or othertissues associated with bone or joints.

[0027] In vitro and in vivo assays for the assessment ofchondroinduction, chondroconduction, osteoinduction and osteoconductionare known by those of ordinary skill in the art. For the in vitro tests,primary fetal rat calvarial cells, harvested by a series of collagenasedigestions, according to the method of Wong and Cohn (PNAS USA72:3167-3171, 1975), or primary rat epiphyseal cartilage Thyberg andMoskalewski, (Cell Tissue Res. 204:77-94, 1979) or rabbit articularchondrocytes, harvested by the method of Blein-Sella 0. et al., (MethodsMol. Biol., 43:169-175, 1995), are seeded into the carriers containingdesired agents and cultured under conventional conditions for 1-4 weeks.Cultures are then processed and evaluated histologically.

[0028] The chondroconductive or chondroinductive capability of a carrierof the present invention containing a desired therapeutic agent can bedetermined by successful support of adhesion, migration, proliferationand differentiation of primary rat bone marrow and stromal cells as wellas primary rat or rabbit chondrocytes. Bone marrow and bone marrowstromal cells are the source of chondroprogenitor cells found in thesubchondral bone marrow of full-thickness defects. Bone marrow can beharvested from the long bones of 2-3 week-old inbred Lewis rats and canbe added directly to a carrier or cultured for 2 weeks under standardconditions. The adherent stromal cell population that grows out of thesecultures is packaged and frozen for use. Cells from up to six passagesare used for culturing or seeding on the carrier to test forchondroconductive or chondroinductive capabilities.

[0029] Retinoic acid-treated chondrocytes represent a less maturechondrocyte and can be used to test the ability of matrices to supportlater stages of chondrogenesis. Retinoic acid treatment of primarychondrocytes is performed prior to culturing or seeding the cells on acarrier (Dietz, U. et al., 1993, J. Cell Biol. 52(1):57-68).

[0030] Cell adhesion and proliferation are monitored using an MTS assaythat can measure cell number and viability based on mitochondrialactivity. Stromal cells or chondrocytes are cultured on a carriercontaining a therapeutic agent for 6-18 hours in the presence or absenceof serum for adhesion analysis and for 1-2 weeks for proliferationassessment.

[0031] For cell migration testing, carriers containing therapeuticagents are coated or fitted onto porous Trans-well membrane cultureinserts (Corning). Stromal cells are seeded on top of the carrier in theupper chamber of the Trans-well and a chemoattractant (growth factor,PDGF) is placed in the bottom chamber. After 12-18 hours of culture thecells that have migrated through the carrier to the bottom side of theTrans-well membrane are quantitated by the MTS assay. The carrier isremoved from the upper chamber and processed histologically to assessthe degree of infiltration.

[0032] The analysis of differentiation markers relevant tochondrogenesis and osteogenesis are evaluated at both the protein andtranscriptional level. The specific markers that may be analyzedinclude: 1) Type II collagen and IIA, IIB isoforms; 2) Aggrecanproteoglycan; 3) Type IX, X and XI collagen; 4) Type I collagen; 5)Cartilage matrix protein (CMP); 6) Cart-1 transcription factor; 7)Fibronectin (EDA, EDB isoforms); 8) Decorin proteoglycan; 9) Linkprotein; 10) NG-2 proteoglycan; 11) Biglycan proteoglycan; 12) Alkalinephosphatase. Differentiation may be measured by Northern/PCR analysis,Western blotting or by metabolic cell labeling.

[0033] For Northern/PCR analysis, RNA is isolated by standard proceduresfrom stromal cells or chondrocytes. Time course tests may be used todetermine optimal culture periods that range from 1 to 6 weeks dependingon the cell type. The isolated RNA is analyzed by Northern gel andhybridization techniques with specific cDNA or PCR amplified probes.Northern analysis is quantified by densitometric scanning ofautoradiographs and normalization to housekeeping gene signals (G3PDH).Northern analysis may be supplemented with quantitative PCR analysisusing primers generated from the published cDNA sequences of the genesto be analyzed.

[0034] For Western blotting, solubilized protein lysates are isolatedfrom cells cultured on carriers containing osteogenic or chondrogenicagents by standard techniques (Spiro R. C., et al., 1991, J. Cell.Biol., 115:1463-1473). After the lysis of cells the carrier is extractedin stronger denaturants (8 M urea, GnHCL) to remove and examine bound orincorporated proteins. Protein samples are analyzed by standard Westernblotting techniques using specific polyclonal or monoclonal antibodies.

[0035] For metabolic cell labeling, cells cultured on a carriercontaining a therapeutic agent are metabolically radiolabeled with³⁵SO₄, ³⁵S-methionine or ³H/¹⁴C-labeled amino acids by standardtechniques (Spiro et al., supra). Solubilized cellular andmatrix-associated proteins are quantitatively immunoprecipitated withantibodies specific for the protein of interest and analyzed by SDS-PAGE(Spiro et al., supra). Quantitation of results are performed bydensitometric scanning of autoradiographs and signals will be normalizedto either cell equivalents or to a house-keeping protein such as actin.

[0036] Additionally, the ability of a carrier of the present inventioncontaining a chrondrogenic agent to support chondrogeneicdifferentiation in vivo may be tested in an inbred rat soft tissueimplant model. Rat bone marrow or stromal cells described above areseeded onto the carrier at high density, cultured overnight in MEMmedium containing 10% FBS serum and antibiotics, then transferred intoMillipore diffusion chambers and implanted intraperitoneally orsubcutaneously into 8 week-old recipients. Chambers are harvested after3 weeks and evaluated histologically for cartilage formation.

[0037] A transplantation model in outbred rats is used to evaluate theability of the carrier containing the chondrogenic agent to maintain thecartilage phenotype in vivo. Rib costal cartilage chondrocytes areseeded onto the carrier at high density and cultured overnight in HamsF-12 containing 1% rat serum and antibiotics. The seeded carriers arethen implanted into posterior tibial muscle pouches created by bluntdissection in 8 week-old male Sprague-Dawley rats. Explants are taken at14 and 28 days and evaluated histologically for compatibility, cartilagegrowth, and maintenance of the differentiated phenotype based onstaining for aggrecan and type II collagen.

[0038] For the in vivo tests, a carrier of the present inventioncontaining an osteogenic agent may be evaluated for the capabilities forsupporting osseous healing in a rat cranial defect model by implantationinto a 5 mm by 3 mm defect created in the parietal bone of 6 weeks oldmale Sprague-Dawley rats. The defects are evaluated at 28 days byradiographic and histologic analysis.

[0039] The in vivo model for cartilage repair is a full-thicknessarticular cartilage defect in the rabbit (Amiel et al., 1985, J. BoneJoint Surg. 67A:91 1). Defects measuring approximately 3.7 mm indiameter and 5 mm deep defect are created in the center of the medialfemoral condyles of adult male New Zealand white rabbits. The defectsare then either filled with carrier containing a chondrogenic agent orleft unfilled as controls. The defects are evaluated morphologically andhistologically at 6 and 12 weeks and then at 6 months and one year.

[0040] The following examples are provided for purposes of illustrationand are not intended to limit the invention in any way.

EXAMPLE I Preparation of Hyaluronate-Amine Derivative

[0041] Free amine groups were introduced to hyaluronate (HA)(LifecoreBiomedical having a molecular weight of 1.3×10⁶) by the reaction ofhyaluronate with ethylenediamine in the presence of water solublecarbodiimide, 1-(3-dimethylaminopropyl)-3-ethycarbodiimimidehydrochloride (EDC). Diamine compounds and EDC (Aldrich) in extremeexcess are required.

[0042] HA 0.4 grams (about 1 mmole of repeat units) in 100 ml PBS (10mM, pH 7) and 6.7 mls of ethylenediamine (100 mmole) were combinedfollowed by adjusting the solution to pH 5.0 using HCI. 4.02 grams ofEDC (210 mmole) was added to the solution and the reaction was allowedto proceed at room temperature for 24 hours and then dialyzed against 4liters of deionized water 4 times for a total of 24 hours, therebyremoving any unreacted ethylenediamine or EDC prior to the cross-linkingreaction so that the cross-linking reaction proceeds without utilizingextraneous cross-linking or ionic binding agents.

[0043] Under this condition, about 20% of carboxyl groups in the sugarchain were converted to amine groups.

EXAMPLE II Preparation of Polysaccharide-Polyaldehyde Derivatives

[0044] HA/polyaldehyde (HA-pAld) was prepared by the oxidation ofhyaluronate using sodium periodate as an oxidizer. HA 1 gram wasdissolved in 80 ml deionized water to which was added 20 ml of 0.5 Msodium periodate. After reaction at room temperature in the absence oflight for 18 hours, glycerol was added to quench the unreactedperiodate, and dialized against a large volume of deionized water. Thedialized HA-pAld solution was lyophilized and the resulting white powderwas stored in the dark at {tilde over (4)}C. Under this condition, about5% of the repeat units in HA were oxidized. The concentration of activealdehyde in the macromolecular chain is controlled by changing theoxidation conditions, for example, the reaction time and amount ofoxidizer.

[0045] Active aldehyde groups carrying chondroitin sulfate(Sigma)(CS-pAld) were prepared by the same method as above.

EXAMPLE III Preparation of HA-NH2/HA-pAld Gel

[0046] 0.2 grams of HA-NH2 and 0.4 grams of HA-pAld were dissolved in 50ml of deionized water separately. Each of the solutions contained 100micromoles of active groups. The two solutions were mixed at roomtemperature under vigorous stirring. A gel formed after 20 minutes. Thegel thus formed was stable in water at a pH range of 0.1 M HCl to 0.1 MNaOH.

EXAMPLE IV Preparation of HA-NH2/HA-pAld Sponge

[0047] The HA-NH2/HA-pAld gel prepared as in Example III was frozen at−7{tilde over (8)} C. and then dried under vacuum at −4{tilde over (0)}C. for 4 hours, −2{tilde over (0)} C. for 8 hours, −{tilde over (4)}° C.for 20 hours, and 1{tilde over (8)} C. for 1 hour.

EXAMPLE V Preparation of HA-NH2/CS-pAld

[0048] The HA-NH2/CS-pAld gel and sponge were prepared by the methodsdisclosed in Example III and IV above except for the substitution ofCS-pAld for HA-pAld.

EXAMPLE VI Preparation of HA-NH2/HA-pAld Carrier Having Crystal VioletEncapsulated

[0049] 2.0 mls of crystal violet solution (1% Sigma) was added to 23 mlsof deionized water containing 0.2 g HA-NH₂ (free amine content, 100micromole). The solution was mixed with 25 mls of HA-pAld solution(aldehyde content, 100 micromole) at room temperature under vigorousstirring. A gel formed after 20 minutes. The gel thus formed wasincubated with 500 ml of deionized water at room temperature, and thewater was sampled and replaced at the time points 1, 2, 4, 6, 8 and 18hours. The crystal violet released from the gel was monitored bymeasuring the O.D. of sampled solutions at 590 nm. The release curve isshown in FIG. 1.

EXAMPLE VII Preparation of HA-NH2/HA-pAld Carrier Having a TherapeuticAgent Immobilized.

[0050] Albumin, bovine-fluorescein isothiocyanate (FITC-BSA, Sigma) waschosen as a model for therapeutic proteins. 10 mgs of FITC-BSA in 2 mlsof deionized water was added to 23 mls of Ha-pAld solution (HA-pAldcontent, 4 g; aldehyde content, 100 micromole). The solution incubatedat room temperature for 20 minutes, then mixed with 25 mls of HA-NH2solution (HA-NH₂ content, 0.2 g; free amine content, 100 micromole)followed by incubation at room temperature for an additional 20 minutes.The gel thus formed was incubated in 500 ml of deionized water at roomtemperature. The incubation medium was replaced at time points 1, 2, 4,6, 8, 24, 48 hours and every two days thereafter for two weeks. Therelease of FITC-BSA in the incubation medium was determined by measuringthe O.D. at 495 nm. As shown in FIG. 2, about 12% of the FITC-BSAreleased from the carrier in the first two hours; after that time nosignificant amount of protein could be found, indicating that theremaining protein was covalently immobilized in the gel.

EXAMPLE VIII Incorporation of Growth Factor Into Matrices

[0051] Basic fibroblast growth factor (bFGF) was incorporated into HAgels either by addition to HA(I) (prepared as in Example I) solutionfollowing mixing with HA(II) (prepared as in Example II) or byincubation of bFGF with HA(I) solution at {tilde over (4)}° C. overnightprior to mixing with HA(I). These two formulations were recorded asHA(I/II) and HA(II/I), respectively. Incubation with HA(II) covalentlylinks the growth factor to the HA via imine bonds. The finalconcentration is: 1 mg of bFGF, 2 mg of HA(I), and 2 mg of HA(II) in 1ml of sucrose buffer without EDTA. Radioactive ¹²⁵I-bFGF was used as atracer for the samples prepared for release kinetics study. Viscoushyaluronate solution (4%, WN) containing bFGF (1 mg/ml) was used ascontrol.

[0052] For an in vivo rat cranial defect assay, growth factorincorporated HA sponge was prepared by diffusion of bone morphogeneticprotein (BMP) into pre-dried HA sponge (5×4×3 mm.L.W.H.) at the rate of30 ig per piece of the sponge followed by lyophilization.

EXAMPLE IX Study of release of growth factor in vitro

[0053] A six well format cell culture insert equipped with PET membranewith the pore size of 0.4 im was used for the in vitro bFGF releasestudy. Sodium citrate buffer (20 mM, pH 5) containing sucrose (9%) andEDTA (1 mM), and DMEM cell culture medium were chosen as release media.Then 40 mg of each sample (HA (I/II), HA (II/I), HA sponge and control,as described in Example VIII) with 2.0 ml of medium were placed in thewells, another 4.0 ml of medium were added to the outside chamber. Theplates were mounted on an orbital shaker platform and shaken at 3{tildeover (7)} C. constantly. The release medium in the outside chamber wascounted for radioactivity by a liquid scitillation counter (Beckman, LS6500) at desired time points and refreshed. About 68, and 90% ofincorporated bFGF were released from HA viscous solution into the DMEMcell culture medium in 4 hours, and 8 hours, respectively. The remainingbFGF was released in one more day. After incubation for 4 hours, 8hours, and 24 hours, about 62, 78, and 88% of the encapsulated bFGF inHA (I/II) was released, respectively. The remaining bFGF was releasedcompletely in another two days. The type of release medium seems to haveno effect on the bFGF release rate. For HA (II/I) gel, only 16, 25, and30% of incorporated bFGF was released from the gel to sucrose bufferafter incubation for 4 hours, 1 day and 2 days, respectively. Theremaining bFGF was released for 2 more weeks. When DMEM cell culturemedium was chosen as the release medium, only 13, 15, and 17% of bFGFreleased from HA (II/I) for the same time period, and 20% of bFGF stillremained in the gels after 2 weeks incubation when the experiment wasterminated.

EXAMPLE X Subperiosteal Injection In Rat Calvaria

[0054] Six week old male Sprague Dawley rats received 50 il injectionsunder the periosteum of the left parietal bone of samples describedbelow. After 14 days, calvaria were harvested and processed forhistological evaluation. The parietal bone thicknesses are given below.Parietal bone thickness, μm (mean ± SD, n = 6) No treatment 259 ± 30 HAgel 276 ± 94 HA (I/II) + 1 mg/ml bFGF 451 ± 97 HA (II/I) + 1 mg/ml bFGF523 ± 81 1 mg/ml bFGF in buffer 350 ± 35 1 mg/ml bFGF in solution HA 281± 30

What is claimed:
 1. A therapeutic composition comprising: abiodegradable carrier, said carrier comprising a first polysaccharidecross-linked to a second polysaccharide, wherein said first and secondpolysaccharides is each a member selected from the group consisting ofhyaluronic acid, dextran, dextran sulfate, chondroitin sulfate, dermatansulfate, keratan sulfate, heparin, heparan sulfate and alginate; andwherein said first and second polysaccharides are covalentlycross-linked to each other through imine bonds between amino groups onsaid second polysaccharide and aldehyde groups from oxidized sugar ringson said first polysaccharide; and a therapeutic agent selected from thegroup consisting of growth factors, cytokines, hormones, DNA constructs,and autologous, allogenic or modified cells.
 2. The composition of claim1, wherein said first polysaccharide is the same as said secondpolysaccharide.
 3. The composition of claim 2, wherein said first andsaid second polysaccharide are both hyaluronate.
 4. The composition ofclaim 1, wherein said first polysaccharide is different from said secondpolysaccharide.
 5. The composition of claim 4, wherein said firstpolysaccharide is hyaluronate and said second polysaccharide ischondroitin sulfate.
 6. The composition of claim 1, wherein said firstpolysaccharide contains an excess of aldehyde groups such that freealdehyde groups remain subsequent to cross-linking to said secondpolysaccharide.
 7. The composition of claim 1, wherein said carrier hasa gel-like form.
 8. The composition of claim 1, wherein said carrier hasa sponge-like form.
 9. The composition of claim 1, wherein saidtherapeutic agent is covalently bonded to said carrier.
 10. Thecomposition of claim 1, wherein said therapeutic agent is entrappedwithin said carrier.
 11. The composition of claim 1, wherein saidtherapeutic agent is a chondrogenic agent.
 12. A method of inducingcartilage growth in vivo, comprising a step of administering acomposition of claim 1 at a site of desired cartilage growth.
 13. Amethod of conducting cartilage growth in vivo, comprising a step ofadministering a composition of claim 1 at a site of desired cartilagegrowth.
 14. A therapeutic composition for supporting cartilage repair,comprising: a biodegradable carrier, said carrier comprising a firstpolysaccharide cross-linked to a second polysaccharide, wherein saidfirst and second polysaccharides is each a member selected from thegroup consisting of hyaluronic acid, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparansulfate and alginate; and wherein said first and second polysaccharidesare covalently cross-linked to each other through oxime bonds betweenamino groups on said second polysaccharide and aldehyde groups fromoxidized sugar rings on said first polysaccharide; a therapeutic agentsupported by the carrier, the therapeutic agent being selected from thegroup consisting of growth factors, cytokines, hormones, DNA constructs,and autologous, allogenic or modified cells; and a population of cellsseeded on or into the carrier.
 15. The composition of claim 14, whereinsaid therapeutic agent is covalently bonded to said carrier.
 16. Thecomposition of claim 14, wherein said therapeutic agent is entrappedwithin said carrier.
 17. The composition of claim 14, wherein saidtherapeutic agent is a chondrogenic agent.
 18. The composition of claim14, wherein said seed cells are chondrocytes.
 19. A method of inducingcartilage growth in vivo, comprising a step of implanting a compositionof claim 14 at a site of desired cartilage growth.
 20. A method ofconducting cartilage growth in vivo, comprising a step of implanting acomposition of claim 14 at a site of desired cartilage growth.
 21. Amethod for preparing a biodegradable device for cartilage repair, saidmethod comprising: preparing a carrier by reacting a firstpolysaccharide derivative having aldehyde groups with a secondpolysaccharide under conditions whereby said aldehyde groups covalentlyreact to cross link with said second polysaccharide to form said carrierand wherein said first and said second polysaccharides are independentlyselected from the group consisting of hyaluronic acid, dextran, dextransulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate,heparin, heparan sulfate and alginate; introducing a therapeutic agentinto or onto the carrier, the therapeutic agent being selected from thegroup consisting of growth factors, cytokines, hormones, DNA constructs,and autologous, allogenic or modified cells; and seeding a population ofcell on or into the carrier.
 22. The method in claim 21, whereinintroducing the therapeutic agent includes mixing the therapeutic agentwith the first polysaccharide derivative or the second polysaccharidederivative before reacting the first polysaccharide derivative with thesecond polysaccharide derivative, such that reacting the firstpolysaccharide derivative with the second polysaccharide derivativeentraps the therapeutic agent within the carrier.
 23. The method inclaim 21, wherein introducing the therapeutic agent includes mixing thetherapeutic agent with the carrier, such that the therapeutic agent isentrapped within the carrier.
 24. The method in claim 21, whereinintroducing the therapeutic agent includes reacting the therapeuticagent with the first polysaccharide derivative or the secondpolysaccharide derivative before reacting the first polysaccharidederivative with the second polysaccharide derivative.
 25. The method inclaim 21, wherein the seeded cells are chondrocytes.
 26. The method inclaim 21, wherein the seeded cells are cultured in the carrier.
 27. Themethod in claim 21, wherein the therapeutic agent is a chondrogenicagent.
 28. A method of supporting cartilage repair in vivo, said methodcomprising: preparing a biodegradable carrier by reacting a firstpolysaccharide derivative having aldehyde groups with a secondpolysaccharide under conditions whereby said aldehyde groups covalentlyreact to cross link with said second polysaccharide to form said carrierand wherein said first and said second polysaccharides are independentlyselected from the group consisting of hyaluronic acid, dextran, dextransulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate,heparin, heparan sulfate and alginate; introducing a therapeutic agentinto or onto the carrier, the therapeutic agent being selected from thegroup consisting of growth factors, cytokines, hormones, DNA constructs,and autologous, allogenic or modified cells; seeding a population ofcells on or into the carrier; and implanting the carrier at a site ofdesired cartilage repair.